Gene expression and production of a desired protein in a heterologous cytoplasmic environment is a great advantage of modern biology. Many vector-host systems have been developed to achieve high yield of protein synthesis.
In particular, many proteins of interests are produced by expression systems as recombinant proteins in order to improve the yield of synthesis, to rationalize the production, to facilitate their production or to limit the risk of contamination by accompanying compounds.
Examples of recombinant biomolecules of interests in biotechnology are antigens, antibodies and fragments thereof for vaccines, enzymes in medicine or agro-food industry, hormones, cytokines or growth factor in medicine or agronomy.
The rapid development of high throughput technologies needs also to provide cells expressing protein or polypeptide of interest, such as an antigen, an antibody, a receptor for identifying ligands, agonists or antagonists thereof.
Synthesis of specific RNAs can also be useful for their subsequent use in protein synthesis, in diagnosis or in anti-sense therapeutic approach for example.
Usual methods of recombinant protein synthesis include in vivo expression of recombinant genes from strong promoters in host cells, such as bacteria, yeast or mammalian cells or in vitro expression from a DNA template in cell-free extracts, such as the S30 system-based method developed by. Zubay (1973), the rabbit reticulocyte system-based method (Pelham and Jackson, 1976, Eur. J. Biochem., 67: 247–256) or wheat germ lysate system-based method (Roberts and Paterson, 1973, PNAS, 70: 2330–2334).
One advantage of a cell-free system is that it allows the synthesis of proteins harmful for host cells (Zubay, 1973;). Cell-free synthesis has been used as an essential component for translation-based screening in polysome display, truncation test, scanning saturation mutagenesis, site-specific incorporation of unnatural amino acids into proteins, stable-isotope labeling of proteins. Both circular and linear exogenous DNAs can be used as templates for protein synthesis and a high yield of protein synthesis can be reached from strong phage promoters in bacterial cell-free system ( ). However, many unknown factors affect the yield and the homogeneity of protein synthesis in a cell-free system and, therefore, a wide application is limited. Therefore, there is a need to improve the available methods for protein or RNA synthesis, especially with regard to the yield of synthesis and the homogeneity of production.
Actually, improvements at the different steps of gene express ion are required to increase the yield of RNA or protein synthesis in an expression system. Nevertheless, the most crucial steps are the steps of transcription initiation and translation initiation. Therefore, the best known expression systems in the art are based on the use of strong transcriptional and translational signals. As an example, strong phage promoters are widely used for gene expression and protein production both in living cells or cell-free extracts (Studier et al., 1990).
If the different components involved in transcription and translation synthesis are well-known in the Art, the specific contribution of each component is still controversial. Since the steps of transcription and translation initiation are considered to be the rate-limiting steps in RNA or protein synthesis, fundamental mechanisms of these cellular process should be better understood. With regards to the step of transcription initiation, the RNA polymerase is the key enzyme either in cellular or in cell free system.
The Escherichia coli RNA polymerase core enzyme is assembled sequentially to a heterotetrapolymer in the following order: α>α2>α2β>α2ββ′. Said core enzyme α2ββ′ acquires the capacity to recognize and bind to specific sequences of promoter only after its association with exchangeable σ subunits, thereby forming the functionally active holoenzyme able to catalyse RNA synthesis (see for reviews, Gross et al., 1992). It has been shown, that the presence of AT-rich sequences located in promoters can increase mRNA synthesis in vitro in the absence of other added factors. Later, it has been revealed that the α subunit binds to a AT-rich sequence, known as a UP element, located upstream a −35 site of strong promoters (Ross et at., 1993). However, UP element appears to be present in other promoters as well (Estrem et al., 1998). The α subunit of the RNA polymerase (encoded by the rpoA gene is composed of two domains, an N-terminal domain (αNTD) and a C-terminal domain (αCTD), which are connected by a flexible protease sensible linker. The αNTD domain is necessary for RNA polymerase assembly whereas the αCTD domain is involved both, in DNA-binding and in specific interactions with a number of transcription factors, including cAMP receptor protein (CRP), OxyR, TyrR, GaIR (Ishihama, 1993). More specifically, Jeon et al., have shown that the same αCTD protein region is involved in binding to UP element and to specific transcription factors (Jeon et al., 1997). Two α subunits bind in tandem to the UP element. The helix IV of the αCTD domain binds directly to some promoters (Ozolino et al., 2000). Analysis of UP elements in a library of random sequences allowed the selection of UP elements increasing in vivo transcription synthesis up to 330-fold (Gourse et al., 2000).
RNA synthesis is decreased in in vitro transcription system in presence of a reconstituted RNA polymerase, wherein the α subunit is mutated for binding to the UP-element as compared to RNA synthesis level obtained with the reconstituted holoenzyme with a wild-type α subunit (Gaal et al., 1996; Ross et al., 1998). However, the overexpressed E. coli RNA polymerase α subunit has been shown to inhibit overexpression of osmoregulatory porin genes ompF and ompC in cells and the purified α subunit added along with RNA polymerase inhibits synthesis from the ompF promoter in in vitro transcription system (Bowrin et al., 1994).
These last results show the major role of UP elements and probably, their interacting proteins, in the modulation of the level of mRNA synthesis in bacterial cells.